In vitro DNA manipulation allows the transfer of foreign genetic information into a host cell to affect efficient expression of endogenous and foreign proteins in a wide variety of host cells, such as microbial hosts. Recombinant DNA techniques have made possible the selection, amplification and manipulation of expression of proteins and peptides.
Some modifications to a recombinantly produced protein or peptide, however, cannot be accomplished by altering the DNA sequence. Many naturally occurring proteins and peptides contain a C-terminal amino acid residue that has an xcex1-carboxamide group but the amide group is not produced directly through expression. Instead, a precursor protein is produced by genetic expression and the amide is introduced in vivo by enzymatic modification of the precursor protein. In vitro, a variety of methods exist for converting a C-terminal xcex1-carboxylic acid group into an xcex1-carboxamide group, however, the available methods generally have limitations in terms of a number of factors, such as the reaction conditions, selectivity, type of reagent(s) employed and/or types of substrates which may be used.
Moreover, many small foreign proteins and oligopeptides often cannot be successfully overproduced in most cellular hosts, since the host may reassimilate the peptide after expression. For example, where the size of the desired peptide is no more than about 60 to 80 amino acid units in length, degradation rather than end product accumulation usually occurs.
In response to this problem, small peptides have typically been expressed either as part of fusion proteins which include a second larger peptide (e.g., xcex2-galactosidase or chloramphenicol acetyl transferase) or as a recombinant construct which includes multiple copies of the desired peptide (a multicopy construct). In either instance, the initially expressed construct generally needs to be cleaved to produce the desired peptide(s). Very often, the recombinant construct is cleaved to produce a precursor peptide(s) which may then be subjected to posttranslational modification to produce the desired peptide(s). It would be extremely advantageous to have additional method(s) which would allow cleavage of a peptide precursor to be carried out simultaneously with the introduction of an xcex1-carboxamide group into the C-terminal amino acid residue of the cleavage product.
The invention relates to a method of producing a polypeptide having a C-terminal xcex1-carboxamide group. It particularly concerns an enzymatic modification of selected arginine-containing substrate polypeptides which result in cleavage of the substrate polypeptide to form a product polypeptide having a C-terminal xcex1-carboxamide group. The method includes contacting a substantially aqueous solution which includes (a) the substrate polypeptide (xe2x80x9cfirst polypeptidexe2x80x9d) and (b) ammonia reagent with (c) clostripain. The substrate polypeptide includes at least one copy of a core amino acid sequence and typically includes more than one copy of the core amino acid sequence (i.e., a multicopy construct). The C-terminal residue of the core amino acid sequence is an arginine residue which is bonded to the adjacent amino acid residue through an xcex1-carboxyl peptide bond (i.e., an xe2x80x9cArg-Xaaxe2x80x9d peptide linkage). Since clostripain is an endopeptidase, the Xaa amino acid residue represents an amino acid residue which has its xcex1-carboxylic group bonded to either another amino acid residue through a peptide bond (xe2x80x9cArg-Xaa-Xaaxe2x80x2xe2x80x9d) or to a carboxyl blocking group (xe2x80x9cArg-Xaa-Rxe2x80x9d). Carboxyl blocking groups are organic functional groups which replace the acid functionality of the carboxylic acid (the xe2x80x9cxe2x80x94OHxe2x80x9d portion of the xe2x80x94C(O)OH group) and are capable of being cleaved or hydrolyzed to regenerate a carboxylic acid group (xe2x80x9cxe2x80x94C(O)OH groupxe2x80x9d). Examples of suitable carboxyl blocking groups include groups include the alkoxy portion of an ester group (e.g., the ethoxy or benzyloxy portion of a xe2x80x94C(O)OR group) and the xe2x80x94NRRxe2x80x2 portion of a non-peptide amide linkage (e.g., the NRRxe2x80x2 portion of a xe2x80x94C(O)NRRxe2x80x2 group). The xe2x80x94NRRxe2x80x2 portion may be unsubstituted (i.e., NH2) or may be substituted with one or two substituents (e.g., NHEt or NMe2). When such a substrate polypeptide in an aqueous-based solution is contacted with the ammonia reagent in the presence of clostripain, the substrate polypeptide is cleaved at the xcex1-carboxyl peptide bond of the arginine residue and a second polypeptide (xe2x80x9cproduct polypeptidexe2x80x9d) having a C-terminal arginine residue containing an xcex1-carboxamide group (xe2x80x9cArg-NH2xe2x80x9d residue) is produced.
As employed herein, the term xe2x80x9cammonia reagentxe2x80x9d refers to a reagent which includes xe2x80x9cdissolved free ammoniaxe2x80x9d (i.e., NH3 dissolved in the aqueous solution) and/or is capable of releasing free dissolved ammonia in an aqueous solution under conditions where clostripain will amidatively cleave an arginine-containing peptide. For example, the ammonia reagent may include one or more salts of ammonia in equilibrium with dissolved free ammonia. The relative amounts of free ammonia and the various salts will generally be a function of various parameters well known to those skilled in the art, such as the pH of the solution, the relative concentrations of different anions present in the solution and/or the solubility of particular individual salts of ammonia. Since the pKa of ammonia (xe2x80x9cNH3xe2x80x9d) is about 9.2 in aqueous solution, a substantial portion of the ammonia reagent will generally be present as free ammonia at pHs of about 9 or above. In solutions with a pH above the pKa of ammonia, more than half of the ammonia will generally be present either as dissolved free ammonia or as ammonium hydroxide (xe2x80x9cNH4OHxe2x80x9d). It also will be understood that the anion portion of a salt of ammonia generally undergoes a very rapid exchange with other anions present in a given solution. Thus, if a pH 10.0 aqueous solution includes chloride salt(s) (xe2x80x9cClxe2x88x92xe2x80x9d), acetate salt(s) (xe2x80x9cOAcxe2x88x92xe2x80x9d) and sulfate salt(s) (xe2x80x9cSO4=xe2x80x9d), ammonia reagent in this solution will likely include ammonium chloride (xe2x80x9cNH4Clxe2x80x9d), ammonium acetate (xe2x80x9cNH4OAcxe2x80x9d) and ammonium sulfate (xe2x80x9c(NH4)2SO4xe2x80x9d), as well as dissolved free ammonia and ammonium hydroxide (xe2x80x9cNH4OHxe2x80x9d). The present method typically employs the aqueous-based reaction medium which includes at least about 0.5 M ammonia reagent. It appears that a concentration of ammonia reagent of about 0.75 M to about 1.5 M strikes a balance between optimizing the rate and yield of amidated product formation while avoiding substantial inhibition of the enzyme activity. As employed herein, the concentration of ammonia reagent is based on the equivalents of free dissolved NH3 that are present in the medium. One embodiment of the present method includes forming a solution of the substrate polypeptide in a first aqueous-based medium having a pH of no more than about 8.5 and, preferably having a substantially neutral pH. The substrate polypeptide may be cleaved at the xcex1-carboxyl peptide bond to produce the product polypeptide having a C-terminal Arg-NH2 residue by adjusting the pH of the solution to at least about 9.0 and, typically between about 9.0 to about 11.0, and contacting the substrate polypeptide with an immobilized form of clostripain (xe2x80x9cimmobilized clostripainxe2x80x9d) in the presence of ammonia reagent. The substrate and ammonia reagent are preferably contacted with the immobilized clostripain for no more than about 20 minutes and, more preferably, for no more than about 5 minutes.
Typically, the first aqueous-based medium is mixed with a basic aqueous solution (xe2x80x9calkaline mediumxe2x80x9d) to raise the pH shortly before the substrate polypeptide and ammonia reagent are brought into contact with the immobilized clostripain. One manner of practicing this embodiment of the invention is to pack resin containing immobilized clostripain in a chromatography column. The substrate stock solution and basic solutions are mixed just prior to introduction to the column, thereby minimizing the exposure of the substrate polypeptide to high pH aqueous solution. In a typical embodiment of the invention, the basic aqueous solution includes the ammonia reagent. This is not required, however, as some or all of the ammonia reagent may also be present in the reaction medium prior to raising the pH of the reaction medium to at least about 9.0.
Generally, it is also preferred to adjust the pH of the reaction mixture to a value below about 8.5, and preferably to a substantially neutral pH (e.g., a pH of about 6.5 to about 8.0) shortly after the product polypeptide is removed from contact with the immobilized enzyme. Typically, the pH of the reaction mixture containing the product polypeptide is adjusted to about 8.5 or below as soon as the mixture exits the column containing the resin bed with immobilized clostripain. This decreases the chances of the product polypeptide being degraded under the relatively high pH aqueous conditions employed for the clostripain catalyzed amidative cleavage. Polypeptides are known to be susceptible to racemization and/or degradation via hydrolysis under high pH aqueous conditions.
It is typically advantageous to choose the conditions under which the substrate polypeptide is contacted with the immobilized clostripain in the presence of ammonia reagent so as to minimize the amount of time that the substrate and product are subjected to high pH conditions. The present method can be conducted in a manner allows a high yield conversion of substrate to amidative cleavage product while limiting the time the substrate/product solution is in contact with the immobilized enzyme at a pH greater than about 8.5 to no more than about 30 minutes. Preferably, the amidative cleavage is conducted in such a manner that the substrate/product solution is at a pH of 8.5 or above for no longer than about 20 minutes and, more preferably, no longer than about 5 minutes (e.g., the cleavage reaction is carried out in about 2-5 minutes).